Magnetic sensor, production method of the same, and target substance detecting apparatus and biosensor kit using the same

ABSTRACT

The present invention provides a magnetic sensor which detects a target substance indirectly by making a labeling substance larger than the target substance bond with the target substance contained in a sample in a detection area, and detecting the labeling substance, comprising a capture area which is relatively easy to capture the target substance, and a non-capture area which is relatively hard to capture the target substance, on a surface of a member which is comprised in a detection area, wherein the capture area is surrounded by the non-capture area. Thereby, the sensor enables to detect comparatively accurately the number and concentration of substances which cannot be directly detected, and enables to be used for detection of various target substances.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a magnetic sensor for target substancedetection and its production method, and an apparatus and a kit fortarget substance detection using this sensor.

2. Description of the Related Art

As a quantitative immunoassay, a radiometric immunity analysis (RIA:radio immunoassay or IRMA: immunoradiometric assay) has been known formany years. This method labels a competitive antigen or an antibody by aradionuclide, and quantitatively measures the antigen from a measurementresult of specific radioactivity. Thus, this method labels a targetsubstance, such as an antigen, and measures this indirectly. Althoughthis method has achieved large contribution in clinical diagnosis sincesensitivity is high, there is a problem of safety of a radionuclide, andthere is a defect that a facility and an apparatus for exclusive use arenecessary. Then, methods of using labels, such as a fluorescentmaterial, an enzyme, an electrochemical luminescence molecule, and amagnetic particle, have been proposed as methods of being easier tohandle, for example. An enzyme immunoassay (EIA) which uses an enzyme asa label is a method of making an antigen-antibody reaction performed,making an enzyme-labeled antibody react, adding a substrate to theenzyme and making it color, and performing colorimetry with itsabsorbance.

Recently, a method of detecting easily a trace amount of magneticparticles used as a labeling substance is proposed by using amagnetoresistance effect film (refer to David R. Baselt et al.,Biosensors & Bioelectronics 13, 731 (1998)) (hereafter, “Document 1”)and D. L. Graham et al., Biosensors & Bioelectronics 18, 483 (2003)(hereafter, “Document 2”).

In Document 1, two or more pieces of magnetic particles with 2.8 μm ofdiameter are detected using giant magnetoresistance effect (GMR) filmsin size of 80 μm×5 μm and 20 μm×5 μm. The magnetic film used in the GMRfilm is an inplane magnetization film, and a magnetic field applied tomagnetic particles is applied in a direction perpendicular to a filmsurface of the magnetic film. Hence, as illustrated in FIG. 4, a straymagnetic field generated by the magnetic particles magnetized by anapplication of a magnetic field is applied to the magnetic film of theGMR film in an approximately in-plane direction, and magnetization ofthe magnetic film is aligned in this magnetic field direction. FIG. 4illustrates a magnetic sensor 200, a magnetic particle 400, amagnetization vector 410, an applied magnetic field vector 420, and astray magnetic field 430. Magnitude of electric resistance of amagnetoresistance effect film depends on a relative magnetizingdirection of two magnetic films. That is, a parallel magnetizingdirection makes electric resistance comparatively small and ananti-parallel magnetizing direction makes electric resistancecomparatively large. In order to achieve magnetized states of theparallel and anti-parallel, the magnetoresistance effect film includesmagnetic materials having such coercive forces that a magnetizingdirection of a magnetic film in two magnetic films of themagnetoresistance effect film is fixed and a magnetizing direction ofanother film is magnetization reversible by a stray magnetic field froma magnetic particle. When a magnetic particle does not exist on a GMRsensor, since a magnetic field in an in-plane direction is not appliedto a magnetic film even if an external magnetic field is applied,magnetization reversal does not occur. A detection circuit hasconstruction that a bridge circuit includes fixed resistors, and twosensors, that is, a GMR sensor in which a magnetic particle is notimmobilized, and a GMR sensor in which a magnetic particle can beimmobilized, and a lock-in amplifier detects potential differenceinduced in this bridge circuit. In Document 2, a magnetic particle in 2μm of diameter is detected using a GMR sensor in the size of 2 μm×6 μm.Similarly to Document 1, the GMR sensor is formed by arranging one inwhich a magnetic particle can be immobilized, and another in which amagnetic particle is not immobilized, and detects the magnetic particleby comparing output signals of these two GMR sensors. Nevertheless, amagnetic film is an inplane magnetization film, and a magnetic fieldapplied to the magnetic particle is in an in-plane longitudinaldirection to the magnetic film.

As described above, detection methods of a magnetic particle using amagnetoresistance effect film perform detection by magnetizing themagnetic particle in a desired direction, and changing a magnetizingdirection of the magnetoresistance effect film by a stray magnetic fieldemitted from the magnetic particle. These methods have advantages thatan apparatus, a reagent, and the like which are used for measurement aresimply handled, and that it is detectable in a comparatively short time.

For example, when a target substance which is going to be detected is anantigen, immobilization of the magnetic particle on the sensor isperformed using an antigen-antibody reaction. That is, a primaryantibody is formed on the sensor, a specimen, such as blood which maycontain an antigen, is made to react with the antibody, and then, themagnetic particle modified by a secondary antibody is made to react.Because of this series of operations, when the antigen exists in thespecimen, bonding of primary antibody-antigen-secondaryantibody-magnetic particle occurs on the sensor. If the antigen does notexist, the above-mentioned bonding cannot be performed and the magneticparticle is not immobilized on the sensor as a result.

Since one magnetic particle is immobilized on the sensor to one targetsubstance when the above-mentioned immobilizing method is used, the onetarget substance is detectable by using the magnetic sensor with highsensitivity.

As labeling substances, various things, such as fluorescent substances,enzymes, electrochemical luminescent substances, radioactive substances,and magnetic substances, exist, and the number and concentration oftarget substances are detected using a detection unit suitable for alabeling substance.

When a fluorescence label, an enzyme label, an electrochemicalluminescence label, or the like is used as a label in an opticalmeasuring method, and detection of a target substance is performed bymeasuring an optical absorbance, a transmittance, or an amount of lightemission. In addition, when using the radioactive label containing aradioactive isotope, specific radioactivity is measured and aquantitation of a target substance is performed. Using the above opticalmeasurement methods and radiation measurement methods has a demerit thata large measuring apparatus is necessary. On the other hand, when amagnetic label proposed recently is used, detection using a smallmeasuring apparatus is possible. In the case of using the magneticlabel, a small magnetic sensor detects a magnetic field generated from amagnetic particle. As the magnetic sensor, a hall device or amagnetoresistive element is usable.

In the sensor in Document 2, a film with single composition is formed onthe sensor, and substances with which target substances bond, forexample, antibodies are arranged uniformly. Although size of a modifiedmagnetic particle used for a biosensor is hundreds of nm to tens of μm,size of substances bonding with target substances, such as antigens, isseveral nm in many cases. Hence, as shown in FIG. 5A, if antibodies andthe like are densely formed on the sensor, even if all the antibodiesare absorbed in antigens, since a magnetic particle is far larger thanan antibody, there arises a case that magnetic particles may not beimmobilized to all the antigens. In consequence, there is a problemthat, since the number of the immobilized magnetic particles isremarkably different from the number of the adsorbing antigens, there isa possibility that it becomes hard to detect the number or concentrationof the antigens. For example, as illustrated in FIG. 5B, even whenantigens are immobilized, the number of magnetic particles is notdifferent from the case illustrated in FIG. 5A although the number ofthe antigens is different, and the number of magnetic particles and thenumber of antigens are not in one-to-one correspondence, and hence, thecorrect number of antigens is not detected.

SUMMARY OF THE INVENTION

The present invention aims at providing a sensor which can solve theproblem of detection sensitivity based on relationship between theabove-mentioned target substance and label.

A magnetic sensor provided by the present invention is a magnetic sensorwhich detects a target substance indirectly by making a labelingsubstance larger than the target substance bond with the targetsubstance contained in a sample in a detection area, and detecting thelabeling substance, and is characterized by having a capture area whichis relatively easy to capture the above-mentioned target substance, anda non-capture area which is hard to capture the above-mentioned targetsubstance, on a surface of a member which constructs the above-mentioneddetection area, and the above-mentioned capture area is surrounded bythe above-mentioned non-capture area.

A first aspect of a production method of a magnetic sensor provided bythe present invention is a production method of a magnetic sensor whichhas a capture area which is relatively easy to capture a targetsubstance, and a non-capture area which surrounds the capture area andwhich is hard to capture the above-mentioned target substance, on asurface of a member which constructs a detection area, and detects theabove-mentioned target substance indirectly by making a labelingsubstance larger than the target substance bond with the targetsubstance contained in a sample in the detection area, and detecting thelabeling substance. The present method is characterized by includingforming an aluminum film on a film which is made of a material whichconstructs the above-mentioned non-capture area, anodizing theabove-mentioned aluminum film to form a hole, filling up theabove-mentioned hole with a material which constructs theabove-mentioned capture area, and removing the above-mentioned aluminumfilm with leaving the above-mentioned filled area and exposing the filmwhich is made of the material which constructs the above-mentionednon-capture area.

Another aspect of the production method of a magnetic sensor provided bythe present invention is a production method of a magnetic sensor whichhas a capture area which is relatively easy to capture a targetsubstance, and a non-capture area which surrounds the capture area andwhich is hard to capture the above-mentioned target substance, on asurface of a member which constructs a detection area, and detects theabove-mentioned target substance indirectly by making a labelingsubstance larger than the target substance bond with the targetsubstance contained in a sample in the detection area, and detecting thelabeling substance. The present method is characterized by includingapproaching a needle, which includes the material which constructs theabove-mentioned capture area, to the film which is made of the materialwhich constructs the above-mentioned non-capture area, applying avoltage between the needle and the films which is made of the materialwhich constructs the above-mentioned non-capture area, and forming adot, which is made of the material which constructs the above-mentionedcapture area, on the film which is made of the material which constructsthe above-mentioned non-capture area.

Further aspect is a production method of a magnetic sensor which has acapture area which is relatively easy to capture a target substance, anda non-capture area which surrounds the capture area and which is hard tocapture the above-mentioned target substance, on a surface of a memberwhich constructs a detection area, and detects the above-mentionedtarget substance indirectly by making a labeling substance larger thanthe target substance bond with the target substance contained in asample in the detection area, and detecting the labeling substance. Thepresent method is characterized by including forming a hole in a resistfilm arranged on a film which is made of a material which constructs theabove-mentioned non-capture area, forming a material, which constructsthe above-mentioned capture area, on the above-mentioned resist film andfilling up the above-mentioned hole with a material which constructs theabove-mentioned capture area, and removing the above-mentioned resistfilm, and exposing the film which is made of a material which constructsthe above-mentioned non-capture area.

Still another aspect is a production method of a magnetic sensor whichhas a capture area which is relatively easy to capture a targetsubstance, and a non-capture area which surrounds the capture area andwhich is hard to capture the above-mentioned target substance, on asurface of a member which constructs a detection area, and detects theabove-mentioned target substance indirectly by making a labelingsubstance larger than the target substance bond with the targetsubstance contained in a sample in the detection area, and detecting thelabeling substance. The present method is characterized by includingforming a film which is made of the material which constructs theabove-mentioned non-capture area, forming a hollow in a surface of theabove-mentioned film, and forming the material which constructs theabove-mentioned capture area in the hollow.

A detecting apparatus provided by the present invention is a detectingapparatus of a target substance including a magnetic sensor for targetsubstance detection, and a detection unit of detecting capture of atarget substance to the above-mentioned sensor based on a signalobtained in the magnetic sensor, and is characterized in that theabove-mentioned magnetic sensor is a sensor having the above-mentionedconstruction, and that the above-mentioned detection unit has a unit ofdetecting capture of the above-mentioned target substance with comparinga signal, obtained in the above-mentioned sensor, with a referencevalue.

A kit for target substance detection provided by the present inventionis characterized by including a sensor, having the above-mentionedconstruction, and the above-mentioned labeling substance.

Further features of the present invention will become apparent from thefollowing description of exemplary embodiments (with reference to theattached drawings).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a conceptual diagram for describing a structural example of asensor of the present invention;

FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H and 2I are conceptual diagramsdescribing an example of production processes of the sensor of thepresent invention;

FIGS. 3A and 3B are conceptual diagrams for describing a structuralexample of the sensor of the present invention;

FIG. 4 is a schematic diagram illustrating a structural example of amagnetic sensor;

FIGS. 5A and 5B are schematic diagrams illustrating a structural exampleof a magnetic sensor; and

FIG. 6 is a conceptual diagram for describing a structural example of asensor of the present invention.

DESCRIPTION OF THE EMBODIMENTS

In view of the problem cited previously, the present invention proposesa magnetic sensor which has structure which lessens difference betweenthe number of labeling substances, and the number of target substances,and which is also suitable for enabling more accurate quantitativedetection.

Specifically, the magnetic sensor according to the present invention hasa capture area which is relatively easy to capture a target substance,and a non-capture area which is hard to capture the target substance, ona surface of a member which constructs a detection area, and thenon-capture area surrounds the capture area. The capture area which isrelatively easy to capture a target substance is formed as asubmicroscopic area in which the target substance labeled with alabeling substance is easy to immobilize. This area may have a targetsubstance capture function in itself, or may be an area which canimmobilize a substance which has a target substance capture function,such as an antibody.

The magnetic sensor according to the present invention solves theproblem that the number of labeling substances to the target substancesis not in one-to-one correspondence, which is mentioned above usingFIGS. 5A and 5B, and can measure an amount of target substancesaccurately with performing one-to-one correspondence of the number oflabeling substances to target substances.

The magnetic sensor according to the present invention prevents a targetsubstance from being captured by a non-capture area since thenon-capture area which is relatively hard to capture the targetsubstance surrounds an area which is relatively easy to capture a targetsubstance. This enables accurate correspondence between the targetsubstances captured by the capture area and the labeling substancesbonded with these, and enables the detection of the number andconcentration of biomolecules, such as antigens and DNA, moreaccurately.

In the present invention, such construction that a non-capture area isdotted with a plurality of capture areas can be also performed.

In addition, size of one capture area is sufficient for capturing onetarget substance, and insufficient for capturing a plurality of targetsubstances.

In addition, a plurality of capture areas can be spaced apart by adistance larger than a diameter of a labeling substance.

In the present invention, a capture area can be an area containing gold,and a non-capture area can be an area containing silicon. Then, the areacontaining silicon can include silicon nitride, silicon oxide, or amixture of them.

Construction of the sensor according to the present invention enables tohave structure suitable for obtaining such construction that a spacewhich a target substance occupies is smaller than a space which alabeling substance occupies, and a plurality of target substance captureareas is spaced apart by a distance larger than the size of a spacewhich a labeling substance occupies. Arrangement of the plurality oftarget substance capture areas in this way enables to detect the numberand concentration of target substances in a sample more accurately.

A magnetic particle and the like can be used as a labeling substance.

As a detection unit combined with the sensor, it is sufficient to selectand use a suitable way in each of a case of detecting a target substancedirectly, and a case of detecting a target substance indirectly using alabel.

When capture of a target substance into a target substance capture areais detected as a magnetic change, the sensor according to the presentinvention can be constructed as a magnetic sensor which can convert amagnetic change into a signal and can output the signal.

The following respective methods can be suitably used for production ofa member which constructs the detection area of the magnetic sensor withthe above-mentioned construction.

A first method has the following steps.

(1) A step of forming an aluminum film on a film which is made of amaterial which constructs a non-capture area.

(2) A step of anodizing the aluminum film to form a hole.

(3) A step of filling up the above-mentioned hole with a material whichconstructs the above-mentioned capture area.

(4) A step of removing the above-mentioned aluminum film with leavingthe above-mentioned filled area, and exposing the film which is made ofthe material which constructs the above-mentioned non-capture area.

In this method, the size of and the gap between respective targetsubstance capture areas can be adjusted by setting conditions of anodicoxidation suitably.

A second method has the following steps.

A step of approaching a needle, which includes a material whichconstructs the above-mentioned capture area, to a film which is made ofa material which constructs a non-capture area, applying a voltagebetween the needle and the films which is made of the material whichconstructs the above-mentioned non-capture area, and forming a dot,which is made of a material which constructs the above-mentioned capturearea, on the film which is made of the material which constructs theabove-mentioned non-capture area.

In this method, a form and size of a dot, and the gap between respectivedots can be adjusted according to a gap between the needle and thecapture area, the material type which constructs the capture area,application conditions of the voltage, positions of the needle and thefilm, which includes the material which constructs the non-capture area,at the time of a voltage application, and the like.

A third method has the following steps.

(1) A step of forming a hole in a resist film arranged on a film whichis made of a material which constructs a non-capture area.

(2) A step of forming a material, which constructs the above-mentionedcapture area, on the above-mentioned resist film, and filling up theabove-mentioned hole with a material which constructs theabove-mentioned capture area.

(3) A step of removing the above-mentioned resist film, and exposing thefilm which is made of the material which constructs the above-mentionednon-capture area.

In this method, positions of (gaps between) the target substance captureareas are determined by the holes provided in the resist film.

A fourth method has the following steps.

(1) A step of forming a film which is made of a material whichconstructs a non-capture area.

(2) A step of forming a hollow in a surface of the above-mentioned film.

(3) A step of forming a material which constructs the above-mentionedcapture area in the above-mentioned hollow.

For formation of the material, which constructs the capture area, in thehollows in this method, a cluster beam method or the like can be usedsuitably.

A material for forming the capture area which is relatively easy tocapture a target substance includes gold.

A material for forming the non-capture area which is relatively hard tocapture a target substance includes a material containing silicon, andmore specifically, silicon nitride and silicon oxide.

A target substance detecting apparatus can be constructed using at leastthe sensor with the above-mentioned construction, and a detection unitof detecting capture of a target substance to the sensor based on asignal obtained from the sensor.

Furthermore, a kit for target substance detection can be provided usingat least the sensor with the above-mentioned construction, and alabeling substance. Moreover, a kit for target substance detection canbe provided using at least the detecting apparatus with theabove-mentioned construction, and a labeling substance. Various kinds ofreagents for preparing a sample and making a sample react with thesensor can be further included in this kit.

Hereafter, the present invention is described in detail below withmaking detection of an antigen in a sample solution, in which a labelingsubstance is a magnetic particle, as an example.

As illustrated in FIG. 1, a magnetic sensor 200 is formed in a housing100, and a magnetic sensor 200 is connected to an external detectioncircuit 300. The magnetic sensor 200 may be any magnetic field detectingelement, such as a magnetoresistive element or a hall device. Amongthem, for detecting a weak stray magnetic field from the magneticparticle 400, what has good sensitivity is suitable. As such an element,a TMR (Tunneling Magnetoresistance) element which is a magnetoresistiveelement, or a BMR (Ballistic Magnetoresistance) element is usedsuitably. In order to insulate a sample 500 and the magnetic sensor 200electrically, an insulating film 600 is formed on the magnetic sensor200. Nevertheless, when there is no necessity of insulating the sample500 from the magnetic sensor 200, the insulating film 600 may not beformed. An immobilization film 700 formed so that an area 710 which iscomparatively easy to immobilize (relatively easy to capture) an antigen820 which is a target substance, and an area 720 which is comparativelyhard to immobilize (relatively hard to capture) the antigen 820 may beintermingled is formed on the insulating film 600. Here, members 710,720, and 700 construct a member which constructs a detection area. Thearea 710 which is comparatively easy to immobilize a target substance isformed by immobilizing to a predetermined area a primary antibody 810which adsorbs specifically to a substance to be detected, that is, theantigen 820 which is a target substance. A state of one primary antibody810 being immobilized in one area is satisfactory, and it iscontrollable by an area of the area 710, concentration of an antibody,an immobilizing process, and the like. Subsequently, the sample 500 iscontacted to the primary antibody 810. When the antigen 820 which is atarget substance exists in the sample 500, the antigen 820 bonds withthe primary antibody 810. Furthermore, when injecting the magneticparticle 400 in which the secondary antibody (antibody bonded in aposition which a primary antibody of an antigen has not bonded) 830specifically bonded with the antigen 820 is modified, the magneticparticle 400 is immobilized in a magnetic detection area through theprimary antibody 810, antigen 820, and secondary antibody 830.

According to the above-mentioned method, since the magnetic particle 400is immobilized to the antigen 820 immobilized to the primary antibody810 at a high rate through the secondary antibody 830, the number andconcentration of the antigens 820 can be detected accurately.

Here, a more suitable embodiment of the present invention will bedescribed using FIG. 6. FIG. 6 illustrates an example that two areas 710which are relatively easy to capture the antigens 820, which are targetsubstances, in the sensor construction illustrated in FIG. 1 arearranged. Thus, this figure is made by illustrating two areas 710 whichare easy to capture an antigen, and extracting the area 720, which isrelatively hard to capture the antigen 820, the primary antibody 810,the secondary antibody 830, and the magnetic particle 400 from FIG. 1.Other construction is the same as that illustrated in FIG. 1. In theexample illustrated in FIG. 6, each of the two areas 710 which are easyto capture an antigen has size that one target substance is captured,but has size that a plurality of capture substances cannot be captured.Furthermore, the two areas 710 which are easy to capture an antigen arespaced apart by a distance larger than a diameter of the magneticparticle 400. Thereby, since the antigen 820 and the magnetic particle400 which is a labeling substance become in one-to-one correspondence,there arises no such drawback that the number of antigens(concentration) cannot be grasped accurately, which is described withreferring to FIG. 5B. Thereby, the number (concentration) of targetsubstances can be measured more accurately.

As the antibody used for the present invention, what are conventionallyused are usable. In addition, similarly, various kinds of things can beused as the secondary antibody made to be immobilized to a magneticparticle. As the sample, what antibodies of such as target substances(protein, nucleic acid, and sugar chain), allergens, bacterias, andviruses can recognize specifically become objects. In addition, thepresent invention can detect any substance so long as it is a substancewhich can immobilize directly or indirectly a magnetic particle, withoutlimiting to detection of a biomolecules.

A kit for target substance detection can be provided using at least thesensor according to the above-mentioned construction, and a labelingsubstance.

EXAMPLES

Hereafter, the present invention will be described in detail with citingexamples.

Example 1

In this example, an example of a production method of a magnetic sensoraccording to the present invention will be described with FIGS. 2A to2I.

On a surface of a silicon wafer 201, a multilayer structure is obtainedby sequentially stacking a Cr film 202 at 20 nm film thickness, a Ptfilm 203 at 20 nm film thickness, an MnIr film 204 at 10 nm filmthickness, an FeCo film 205 at 5 nm film thickness, an Al₂O₃ film 206 at1.6 nm film thickness, an FeCo film 207 at 5 nm film thickness, an NiFefilm 208 at 20 nm film thickness, and a Pt film 209 at 5 nm filmthickness by magnetron sputtering (FIG. 2A). As for the Al₂O₃ film 206,oxygen deficiency is compensated by forming a film using an Al₂O₃target, introducing an O₂ gas in a film formation chamber, andperforming plasma oxidation. Starting from a silicon wafer 201 side, theCr/Pt multilayer film becomes a base electrode of a TMR element, theMnlr/FeCo multilayer film becomes a pin layer (layer whose magnetizingdirection is fixed) of a TMR film, the Al₂O₃ film 206 becomes a tunnelfilm of the TMR film, the FeCo/NiFe multilayer film becomes a free layer(layer whose magnetizing direction changes with an applied magneticfield easily) of the TMR film, and the Pt film 203 becomes a protectivelayer for preventing deterioration of the magnetic film at the time ofprocessing.

After coating a resist film at about 1-μm uniform film thickness by aspin coater on a surface of the multilayer film and baking themultilayer structure, patterning is performed into a desired form withultraviolet rays. Then, by performing the baking again, immersing themultilayer structure in a developing solution, and cleaning themultilayer structure with deionized water, a resist film 210 with adesired pattern shape is obtained. Let the resist pattern form be acircular form of 30 μm diameter in this example (FIG. 2B).Publicly-known materials can be used as the material for resistpatterning, such as developing solutions for the resist film, andpatterning processing.

Subsequently, etching from a surface of the multilayer film to a surfaceof the Al₂O₃ film 206 is performed by dry etching using an Ar gas (FIG.2C). Furthermore, an inter-layer insulating film 220 which includesAl₂O₃ is formed by introducing a mixed gas of Ar and O₂, and performingmagnetron sputtering using an Al₂O₃ target (FIG. 2D). Then, the resistfilm and the Al₂O₃ film formed on the resist film are removed bycleaning the multi-layer structure ultrasonically with immersing themulti-layer structure into a resist remover.

On a surface of the multi-layer structure in which the fine patterningwas performed, further, a Pt film 231 at 30 nm film thickness, an Al₂O₃film 232 at 10 nm film thickness, an SiN film 233 at 30 nm filmthickness, a Cr film 234 at 5 nm film thickness, and an Al film 235 at5-μm film thickness are sequentially formed (FIG. 2E). The Pt film 231in this film formation is a top electrode of the TMR element, and theAl₂O₃ film 232 is an insulating film between the TMR element and themultilayer film on the element. A distance between a target substanceand a magnetic sensor is important in sensitivity, and the shorter thedistance is, the higher the magnetic sensor is expected to be insensitivity. Hence, the thinner the multilayer film formed on the freelayer of the TMR film is in a range where each film plays its function,the better the multilayer film is, and hence, each film thickness is notlimited to the value illustrated in this example.

Then, a needle tip with a small radius of curvature at an end is pressedagainst the Al film surface, and hollows 240 are formed (FIG. 2F). Inthis example, the needle whose radius of curvature is about 10 nm isused. In addition, let a gap between adjacent needle tips be 500 nm.

The multi-layer structure on a surface of which the hollows 240 areformed is immersed into an oxalic acid solution, and the Al film isanodized. At this time, anodic oxidation advances with making theportions, in which the hollows are formed in the Al film, as startingpoints. The anodic oxidation is stopped when holes formed reach the Crfilm. An average diameter of the holes formed by the anodic oxidation isabout 15 nm (FIG. 2G).

After cleaning a surface of a multilayer film structure side which hasthe holes which are given anodizing, Au dots 251 are formed on bottomsof the holes formed in the Al film (FIG. 2H). Since aspect ratios ofthese holes are remarkable high, it is suitable that Au atoms comeflying at a vertically high rate to the surface of the multilayer filmstructure. Hence, low pressure remote sputtering, SIS (Self IonizedSputtering), collimate sputtering, ionized sputtering, and the like aregenerally known as such film formation methods.

After film formation of Au is completed, wet etching of the Al film isperformed with an alkali solution. When the Au film formed on the Alfilm becomes an overcoat at this time and etching does not advance,etching should be performed after removing the Au film on the surface ofthe Al film by polishing. After removing the Al film, dry etching of theCr film is performed using a chlorinated gas (FIG. 2I). Nevertheless,since it is necessary to leave the Cr films which exist under the Audots, it is suitable to make chlorine ions collide with Cr surfaces athigh speed, and to make bottoms of the Au dots not etched as much aspossible.

Owing to the above-mentioned process, the magnetic sensor of the presentinvention in which the minute Au dots which dot the SiN film in a 500 nmpitch are formed on the TMR element can be produced.

Although a TMR element is used as a magnetic sensor in this example,various magnetic sensors, such as a GMR element, a BMR element, a Hallelement, an abnormal Hall element, and a planer Hall element, areusable.

The number of magnetic particles in liquid can be detected using themagnetic sensor of the present invention.

A detection circuit is connected between the top electrode and baseelectrode of the magnetic sensor of the present invention, and initialresistance before measurement is measured. When a magnetic particlewhich is an object for detection is remarkably small, and superparamagnetism is exhibited, detection is performed by applying amagnetic field to the magnetic particle from the external. Hence, alsowhen measuring the initial resistance, similarly, a magnetic field isapplied. Magnitude of the applied magnetic field is magnitude which cangive a stray magnetic field of magnitude, by which the magnetic particlecan be detected, to the magnetic sensor. Although the magnitude is notspecified strictly in particular, in this example, the magnetic field ismade to be the magnetic field of 500 Oe perpendicular to the magneticsensor surface. In addition, the detection circuit may be such a highlysensitive detection circuit using a bridge circuit or a lock-inamplifier which is described in Document 1, or may be a circuit withsimple construction which includes a current generator and a voltmeter.

Subsequently, the magnetic sensor is immersed into a sample solutionincluding a magnetic particle as a target substance. The magneticparticle which is an object for detection is immobilized on a surface ofthe magnetic sensor by bonding of the Au dot on the surface of themagnetic sensor, and a thiol group modified by the magnetic particle.Since the magnetic particles which are floating in the sample solutionare gradually immobilized while time passes, the number of the magneticparticles can be known by comparing the detection signal of the magneticsensor at the time when all magnetic particles are immobilized, and theinitial value. Alternatively, even before all magnetic particles areimmobilized, the number of all magnetic particles can be known byestimating a saturation value of the detection signal from the change ofthe detection signal to the immobilization time. When many magneticparticles exist in a sample solution and all magnetic particles cannotbe immobilized on the magnetic sensor, what is necessary is just toimmerse many magnetic sensors into the sample solution.

Furthermore, bio sensing can be performed using the magnetic sensor ofthe present invention. Example of the sensing will be described furtherwith reference to FIG. 1. In addition, the Au dots 251 in theconstruction of FIGS. 2A to 2I are equivalent to the area 710 in FIG. 1which is comparatively easy to immobilize target substances, the surfaceof the SiN film 233 is equivalent to the area 720 which is comparativelyhard to immobilize target substances, and the immobilization film 700 isformed of these. Furthermore, the magnetic sensor obtained through thesteps of FIGS. 2A to 2I is illustrated as reference numeral 200 in FIG.1.

After hydrophilization treatment is performed first, amino silanecoupling agent treatment of the surface of the immobilization film 700is performed. Furthermore, a primary antibody 180 which captures adesired antigen by making an amino group derived from an amino silanecoupling agent, and peptide chains chemically bonded using a crosslinking agent, such as glutaraldehyde, for making the primary antibody810 immobilized is immobilized.

Detection of a prostatic specific antigen (PSA) known as a marker ofprostate cancer according to the following protocol can be tried usingthis detection device. Size of the PSA is as small as about several nm.A primary antibody 810 which recognizes the PSA is immobilized in thedetection device.

(1) Dip the detection device in a phosphate buffered saline includingPSA which is an antigen 820, and incubate for 5 minutes.

(2) Clean the detection device, which passed through step (1), withphosphate buffered saline, and flush the unreacting PSA.

(3) Dip the detection device, which passed through step (2), in aphosphate buffered saline including an anti-PSA antibody labeled by themagnetic particle 400, and incubate for 5 minutes.

(4) Clean the detection device, which passed through step (3), withphosphate buffered saline, and flush the labeled antibody unreacting.

Nevertheless, an average diameter of the magnetic particles 400 is about400 nm, and the magnetic particles 400 exhibit super paramagnetism.Since the immobilization films 700 are formed in the interval of about500 nm, the antigens 820 are not immobilized in an area where themagnetic particles 400 cause steric hindrance.

First, a constant current is flowed through the TMR film in a TMRelement 200 which is a magnetic sensor element in a non-magnetic field,and a voltage of the TMR film at this time is measured. Subsequently, adownward external magnetic field of magnitude of 500 Oe is applied tothe magnetic particle 400 immobilized through the antigen-antibodyreaction on a surface of the TMR film to orient magnetization of themagnetic particle 400 downward. From the magnetic particle 400, a straymagnetic field is generated, a synthetic magnetic field of the externalmagnetic field and the stray magnetic field is applied to the freelayer, and magnetization reversal occurs. In this state, a constantcurrent is flowed again through the TMR film, a variation of the voltageis measured, and the antigens 820 can be detected in the samplesolution.

Example 2

Although the production method of the Au dot pattern using anodicoxidation was described in the first example, Au dots can be also formedinto a desired pattern using an Au target which has a needle-shapedsection.

An SiN film 233 at 30 nm film thickness is formed on a surface of themultilayer film structure in the state that the magnetic sensor of FIG.2D in the first example is formed. Then, a resist film is coated atabout 1-μm uniform film thickness by a spin coater. Subsequently,patterning is performed with ultraviolet rays after baking, for example,so that a resist film may cover an area where Au dots are not formed,such as an area other than an area above the sensor element. Then, themultilayer film structure is baked again, and is immersed in adeveloping solution to be cleaned with deionized water, and then, aresist film with a desired pattern shape is obtained. Next, an acicularAu target and a treated surface are arranged with making the acicular Autarget and the treated surface face and approach each other in parallel.When sputtering of the Au target is performed in this state, Au atomsfrom Au of a needle tip come flying to an element surface, and an Au dotis formed only directly under the needle tip. Then, the element isultrasonically cleaned with being immersed in a resist remover. Theresist film and the Au dots formed on it are removed, and the sensor fortarget substance detection which has a magnetic sensor is obtained. Whena target substance is indirectly detected using a labeling substance,the acicular target where needles are arranged at the pitch larger thana grain size of the labeling substance is used so that the labelingsubstances may be immobilized to the target substances immobilized onthe magnetic sensor 200.

In addition, publicly known materials and methods can be used forformation and removal of the pattern which includes the resist film.

On the other hand, the acicular Au target may be a target that theentire target is formed from Au, or may also have Au portions, such as atarget that an Au film is provided on an acicular base surface.

Example 3

Furthermore, by forming micropores in an upper portion of the element byresist film patterning, and forming Au films on the pores, Au dots canbe also formed into a desired pattern.

An SiN film 233 at 30 nm film thickness is formed on a surface of themultilayer film structure in the state that the magnetic sensor of FIG.2D in the first example is formed. Then, a resist film is coated atabout 200 nm uniform film thickness by a spin coater. After baking,holes are formed by radiating electron rays in positions where Au dotsare formed. Then, the multilayer film structure is baked again, and isimmersed in a developing solution to be cleaned with deionized water,and then, a resist film with a desired pattern shape is obtained.Diameter of the holes formed is about φ50 nm diameter, and the intervalbetween adjacent holes is made larger than the grain size of labelingsubstances. Subsequently, an Au film is formed in each hole by a lowdirective film formation apparatus, and an Au dot that a film thicknessof a center portion is thick is formed in a bottom of each hole. Then,the element is ultrasonically cleaned with being immersed in a resistremover. The resist film and the Au dots formed on it are removed. Inaddition, publicly known materials and methods can be used for formationand removal of the pattern which includes the resist film.

Furthermore, an entire surface of the multilayer film structure isdry-etched with an Ar gas, and a diameter of each Au dot is made smallto desired size. At this time, in order to prevent liberation of N fromthe SiN film, a mixed gas of Ar and N₂ may be used as the etching gas.The sensor for target substance detection which contains the magneticsensor is obtained through the above process.

Example 4

In the third example, a comparatively low dose amount of electron raysare radiated on the resist film coated on the SiN film 233 at 30 nm filmthickness. Then, the multilayer film structure is baked again, and isimmersed in a developing solution to be cleaned with deionized water,and then, a resist pattern as illustrated in FIG. 3A is formed. Thepitch of hollows in the resist pattern is made larger than the grainsize of labeling substances. Then, the resist film is successively madethin by dry etching, and when the lower SiN film 233 is slightly etched,the etching is stopped. After removing the resist film, by flying Auatoms on a surface of the SiN film 233 using a cluster beam, Au atomsgather in the hollows of the SiN film 233, and the Au dots 251 asillustrated in FIG. 3B are formed. The sensor for target substancedetection which contains the magnetic sensor is obtained through theabove process.

In addition, publicly known materials and methods can be used forformation and removal of the pattern which includes the resist film.

According to the exemplary examples of the present invention, a sensorwhich can detect comparatively accurately the number and concentrationof substances which cannot be directly detected, and which can be usedfor detection of various target substances can be provided.

While the present invention has been described with reference toexemplary embodiments, it is to be understood that the invention is notlimited to the disclosed exemplary embodiments. The scope of thefollowing claims is to be accorded the broadest interpretation so as toencompass all such modifications and equivalent structures andfunctions.

This application claims the benefit of Japanese Patent Application No.2006-108498, filed Apr. 11, 2006, which is hereby incorporated byreference herein in its entirety.

1. A magnetic sensor which detects a target substance indirectly bymaking a labeling substance larger than the target substance bond withthe target substance in a sample in a detection area, and detecting thelabeling substance, comprising a capture area which is relatively easyto capture the target substance and a non-capture area which isrelatively hard to capture the target substance on a surface of a memberwhich is comprised in the detection area, wherein the capture area issurrounded by the non-capture area.
 2. The magnetic sensor according toclaim 1, wherein the non-capture area is dotted with the capture areas.3. The magnetic sensor according to claim 2, wherein size of the capturearea is sufficient for capturing the target substance, and insufficientfor capturing a plurality of the target substances.
 4. The magneticsensor according to claim 3, wherein the capture areas are a distancelarger than a diameter of the labeling substance apart.
 5. The magneticsensor according to claim 1, wherein the capture area is an areacontaining gold, and the non-capture area is an area containing silicon.6. The magnetic sensor according to claim 5, wherein the area containingsilicon is comprised of either silicon nitride or silicon oxide.
 7. Amethod for fabricating a magnetic sensor which has a capture area whichis relatively easy to capture a target substance, and a non-capture areawhich surrounds the capture area and which is relatively hard to capturethe target substance, on a surface of a member which is comprised in adetection area, and detects the target substance indirectly by making alabeling substance larger than the target substance bond with the targetsubstance in a sample in the detection area, and detecting the labelingsubstance, comprising the steps of: forming an aluminum film on a filmcomprised of a material which is comprised in the non-capture area;anodizing the aluminum film to form a hole; filling up the hole with amaterial which is comprised in the capture area; and removing thealuminum film with leaving the filled area, and exposing the filmcomprised of the material which is comprised in the non-capture area. 8.A method for fabricating a magnetic sensor which has a capture areawhich is relatively easy to capture a target substance and a non-capturearea which surrounds the capture area and which is relatively hard tocapture the target substance on a surface of a member which is comprisedin a detection area, and detects the target substance indirectly bymaking a labeling substance larger than the target substance bond withthe target substance in a sample in the detection area, and detectingthe labeling substance, comprising the steps of: approaching a needlecomprised of a material which is comprised in the capture area, to afilm comprised of the material which is comprised in the non-capturearea; applying a voltage between the needle and the films; and forming adot, comprised of the material which is comprised in the capture area onthe film comprised of the material which is comprised in the non-capturearea.
 9. A method for fabricating a magnetic sensor which has a capturearea which is relatively easy to capture a target substance and anon-capture area which surrounds the capture area and which isrelatively hard to capture the target substance on a surface of a memberwhich is comprised in a detection area, and detects the target substanceindirectly by making a labeling substance larger than the targetsubstance bond with the target substance in a sample in the detectionarea, and detecting the labeling substance, comprising the steps of:forming a hole in a resist film arranged on a film comprised of amaterial which is comprised in the non-capture area; forming a materialwhich is comprised in the capture area on the resist film to fill up thehole with the material which is comprised in the capture area; andremoving the resist film to expose the film comprised of the materialwhich is comprised in the non-capture area.
 10. A method for fabricatinga magnetic sensor which has a capture area which is relatively easy tocapture a target substance and a non-capture area which surrounds thecapture area and which is relatively hard to capture the targetsubstance on a surface of a member which is comprised in a detectionarea, and detects the target substance indirectly by making a labelingsubstance larger than the target substance bond with the targetsubstance in a sample in the detection area, and detecting the labelingsubstance, comprising the steps of: forming a film comprised of thematerial which is comprised in the non-capture area; forming a hollow inthe surface of the film; and forming a material which is comprised inthe capture area on the hollow.
 11. A detecting apparatus for detectinga target substance which comprises a magnetic sensor for detecting thetarget substance, and a detection unit for detecting a capture of thetarget substance on the magnetic sensor based on a signal obtained inthe magnetic sensor, wherein the magnetic sensor is a sensor accordingto claim 1; and wherein the detection unit has a unit of detectingcapture of the target substance by comparing a signal obtained in themagnetic sensor with a reference value.
 12. A kit for target substancedetection, comprising a magnetic sensor according to claim 1 and alabeling substance.